DESCRIPTION: The purpose of this work is to extend the biomedical scope of high precision compound-specific isotope ratio analysis (CSIA) through an integrated approach involving instrument and methods development side-by-side with biomedical applications. The specific aims build on the progress of the first 3.5 years of the grant, and are as follows. 1) Design and construction of a system to extend the technique of CSIA to measurement of deuterium for both gas chromatography and liquid chromatography; 2) Design and construction of a system for determination of intramolecular isotope ratios, termed "position specific isotope analysis" (PSIA), with complete evaluation of the effectiveness of the proposed strategy and its application to selected biomolecules; 3) Implementation of a novel stable-isotope-based strategy for detection of unknown metabolites of endogenous compounds, particularly fatty acids; 4) Development of an interface for deuterium determination for samples analyzed by a conventional elemental analyzer; 5) Extension of data analysis methods novel to isotope analysis, specifically curve-fitting, to improvement of analytical figures-of-merit, and finally 6) Continued work in human fatty acid metabolism, incorporating analytical innovations into analyses while providing genuine biomedical data upon which to base further analytical refinements. The use of D-labeled tracers along with continuous flow IRMS will facilitate the extension of IRMS to a broader range of studies than currently possible as well as open new applications with dual labels. The first instrument for high precision determination of intramolecular isotope ratios is expected to facilitate tracer studies requiring isotopomer distributions, at 100-fold lower enrichment levels than currently available. In addition, it is expected to permit biomedical scientists to derive information from kinetic isotope effects associated with normal enzyme and non-enzyme meditated metabolism. The proposed approach draws off published pyrolysis studies and recent preliminary data that show predictable fragmentation of biologically relevant molecules. A high sensitivity stable isotope based methodology will help reduce the reliance on radiotracers while providing a straightforward strategy for detecting metabolic changes without extensive knowledge of the possible metabolic pathways involved. A system for high precision D/H analysis for elemental analyzers will facilitate tracer studies in solid samples analyzed routinely with this technique. The final specific aims support previous and planned work. Curve-fitting techniques have been shown to improve the precision and accuracy of a wide range of analyses and so automated routines for data reduction by this method will be written. Finally, continued studies of fatty acid metabolism in humans, particularly transformations of fatty acids into other bioactive molecules, allows timely evaluation and refinement of developed instruments and methods.